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doi: 10.17113/ftb.54.01.16.3973 original scientific paper

Antibacterial Activity of Indian Borage (Plectranthus amboinicus Benth)

Leaf Extracts in Food Systems and Against Natural Microflora in Chicken
Meat
Running Title: Antibacterial Activity of Indian Borage in Food Matrices

Sandeep Kumar Gupta and Pradeep Singh Negi*

Fruit and Vegetable Technology Department, CSIR-Central Food Technological Research Institute,
Mysore -570020, India

Received: November 4, 2014

Accepted: October 21, 2015
Summary

The acetone and ethyl acetate extracts obtained by individual extraction of the leaves
of an Indian traditional medicinal plant, Indian borage (Plectranthus amboinicus Benth) were
evaluated for their ability to prevent spoilage of artificially inoculated model food systems
(cabbage and papaya) and natural microflora of chicken meat. These extracts were able to
reduce the bacterial counts in all food systems, however, the effective concentration varied
with the complexity of the system (cabbage ˂ papaya < chicken). A probable mode of action
of extracts was investigated by analyzing the changes caused by them in bacterial cell wall
and leakage of nucleic acid from bacterial cells. The individual acetone and ethyl acetate
extracts at their respective minimum inhibitory concentration resulted in leakage of cell
constituents to an extent of 40-80% and 60-95%, respectively, as against that of control and
finally leading to disintegration of cell walls. These findings indicate the potentiality of ethyl
acetate and acetone extracts of the leaves of Indian borage for use in food preservation.

Key words: Antibacterial activity, cell constituent leakage, cell wall damage, food model
system, food safety, Plectranthus amboinicus

Introduction

*Corresponding author: Phone: +91-821-2515653; Fax: +91-821-2517233; Email: psnegi@cftri.res.in 1

 Indian borage (Plectranthus amboinicus Benth), known as country borage in English,
is a medicinal plant used widely in Indian system of medicine. It belongs to the family
Lamiaceae (1). It is a succulent, aromatic, perennial herb and decoction of its leaves is used
for several medicinal purposes (2). The antimutagenic, antitumorogenic and antigenotoxic
effects of Indian borage leaves are well documented (3) and recently its antimicrobial
properties also have been reported (4, 5, 6).

Increase in consumer demand for safe and non-synthetic alternatives of chemical

preservatives have directed research interest in exploring extracts of various plant materials
and several essential oils are being explored for their biological potency to delay or inhibit the
growth of pathogenic and/or toxin producing microorganisms in food (7, 8). Several plant
extracts are found to be useful to act as antimicrobial against spoilage and pathogenic
microorganisms. However, their activity decreases considerably when added to a food system
(8). Pomegranate peel extracts was reported to increase the storage life of chicken meat (9)
and grape seed and pine bark extracts were successfully used to control the growth of
artificially inoculated bacteria in raw ground beef (10). Extracts from cinnamon bark,
oregano, clove, pomegranate peel and grape seeds were also reported to decrease various
microbial contaminants in raw pork during storage at ambient temperature (11).

Various antimicrobial mechanisms for natural preservatives have been hypothesized.

Damage to cytoplasmic membrane and membrane proteins, leakage of intracellular contents,
coagulation of cytoplasm, degradation of the cell wall, alteration of genetic material and
depletion of proton motif force are proposed mechanisms of growth inhibition of
microorganisms (7, 8, 12). Dillenia indica extracts was reported to cause disintegration of
cell wall and leakage of genetic material (13). Increase in membrane permeability and leakage
of cytoplasm of bacterial cells by cinnamaldehyde treatment was documented, wherein
cinnamaldehyde interacts with cell wall enzymes (14, 15). Phenolic compounds were reported
to be the major constituent of various plant extracts, and bacterial cell wall lysis after
treatment with phenolics was the main cause of cell death (16).
Although there are numerous studies on antibacterial properties of Indian borage leaf,
they mainly focus on methanol extracts. In our earlier study, we found that acetone and ethyl
acetate extracts in sequential extraction showed higher activity than methanol extract, and
both these extracts showed potential antibacterial activity in laboratory medium when
extracted in individual extraction also (4, 17). Therefore, in the present study, we have used
acetone and ethyl acetate extracts obtained by individual extraction of the leaves of Indian

2
borage to study their antibacterial efficacy in food systems and chicken meat, in order to
evaluate their antibacterial potency under realistic conditions and establish their usefulness as
food preservative. We have also studied the effect of these extracts on bacterial cell wall and
leakage of cell constituents to find out their probable antibacterial mechanism.

Materials and Methods

Plant material and chemical
The leaves of P. amboinicus were collected from the campus of CSIR-Central Food
Technological Research Institute (CFTRI), Mysore, India. The plant material was
identified by Mr. A. S. Chauhan, Principal Scientist, Fruit and Vegetable Technology
Department, CFTRI, Mysore, and a specimen voucher was deposited in the Fruit and
Vegetable Technology Departmental herbarium (FVT DH No. LGMGHCC-PA-8/2011). All
the solvents and chemicals used were of AR grade (Qualigen, Mumbai, India) and
microbiological media and sterilized plates were from HIMEDIA (Mumbai, India).

Plant extract preparation

The fresh leaves of P. amboinicus were washed in running tap water and dried at 55°C
in a hot air oven (Industrial and Laboratory Instrument Corporation, Chennai, India). The
dried leaves were powdered using a mixer grinder (Johnson, Mumbai, India) and extracted
with ethyl acetate and acetone (1:4, leaf: solvent) individually to obtain antibacterial rich
fraction. Flasks were placed in a shaker (60 rpm) for 24 h at ambient temperature. After 24 h,
the extract was filtered with Whatman No 1 filter paper and the filtrate was concentrated in a
fume hood. The chemical profiling of these extracts was undertaken in our earlier study (17).
The total phenolics in the acetone extract (85.15 mg GAE/g extract) were higher than ethyl
acetate extract (67.83 mg GAE/g extract), and the major phenolics present (%) in acetone and
ethyl acetate extract were quercetin (0.1933 and 0.1418), rutin (0.0283 and 0.0207), coumaric
acid (0.0137 and 0.0097), caffeic acid (0.0068 and 0.0054) and gallic acid (0.0033 and
0.0012), respectively.

Microorganisms and minimum inhibitory concentration (MIC) determination

Bacterial strains, namely, Bacillus cereus F 4810 (Public Health Laboratory, London,
UK), Staphylococcus aureus FRI 722 (Public Health Laboratory, The Netherlands),
Escherichia coli MTCC 108 (Microbial Type Culture Collection, Institute of Microbial

3
Technology, Chandigarh, India) and Yersinia enterocolitica MTCC 859 (Microbial Type
Culture Collection, Institute of Microbial Technology, Chandigarh, India) were grown
overnight in brain heart infusion (BHI) broth at 37 C. The MIC for the extracts was
determined by agar dilution method (in-vitro) in our earlier study (17), and the values were
0.0625, 0.075, 0.0625 and 0.1125% for ethyl acetate extract and 0.0375, 0.075, 0.0625, and
0.1 % for acetone extract against B. cereus, S. aureus, E. coli and Y. enterocolitica,
respectively.

Preparation of inoculum
Overnight cultures of B. cereus, Y. enterocolitica, E. coli and S. aureus were harvested
by centrifugation at 5000 rpm for 10 min. The pellet was washed with 2 ml phosphate buffer
saline (PBS, pH 7.4) and was again centrifuged to retain the pellet. The pellet was
resuspended in 1 ml of PBS. Aliquot of 30 μl of the cell suspension was taken and made upto
3 ml using PBS. The absorbance of the suspension was read at 620 nm in a UV-Visible
spectrophotometer (Shimadzu, Japan) and adjusted to ~0.3 (108 CFU/ ml) with PBS in order
to maintain uniformity in the different sets of experiments.

Application in cabbage food model system

Cabbage food model was prepared by adding finely shredded cabbage to the sterile
deionised water (1: 2, w/v) and ground in a mixer for 2-3 min (18). The suspension was
filtered using muslin cloth and pH was adjusted to 7.2 using dilute NaOH/ oxalic acid. The
juice (50 ml) was dispensed in 250 ml conical flask and autoclaved at 121 oC for 20 min.
After cooling, Indian borage extracts were added at different concentrations (MIC, 2xMIC),
and it was inoculated with each bacterium separately to obtain a cell density of 105 CFU/ ml.
Simultaneously, a control without extracts was also used and samples were incubated at 37 oC
for 30 days. The juice was withdrawn at regular intervals and appropriate dilution was pour
plated onto nutrient agar plates. The colonies developed after incubation at 37 oC for 24 h
were counted and the count was expressed as log CFU/ ml.

Application in papaya food system

After washing with tap water, the exterior surface of the ripe papaya fruit was cotton
scrubbed with an alcoholic solution of iodine (2 %, w/v) and allowed to air dry inside a
laminar airflow cabinet. The fruit pulp without seeds (pH 5.3) was aseptically removed with
sterilized spoon, transferred to a sterilized flask and homogenized using the spoon. Pulp (50

4
g) was transferred to sterilized flasks and it was pasteurized at 80 oC for 1 min in order to
eliminate the background microflora (19). After cooling, the Indian borage extracts were
added at different concentrations (MIC, 2xMIC, 5xMIC) and it was inoculated with each
bacterium separately to obtain cell density of 105 CFU/g. Simultaneously, a control (without
extracts) was also used and all the samples were incubated at 37 oC for 7 days. The pulp was
withdrawn at regular intervals and plated using appropriate dilution onto nutrient agar plates.
The colonies developed after incubation at 37 oC for 24 h were counted and the count was
expressed as log CFU/g.

Application in chicken meat system

Chicken meat system was prepared as described by Shan et al. (11). Fresh chicken
meat purchased from local market of Mysore was packed in pre-sterilized polyethylene covers
and transported immediately in an Ice bucket to laboratory. Excess fat and accessory muscles
on the surface were trimmed with a sanitized knife under aseptic conditions. The meat was cut
into pieces of uniform size and placed in a sterile conical flask. Plant extracts (individual
acetone and ethyl acetate) were added at different concentrations (5xMIC, 10xMIC), mixed
thoroughly and incubated for 7 days at 37 ºC. Chicken samples without plant extracts were
used as control. Sampling was done at regular intervals and natural microflora of chicken
meat sample was counted by pour plating of appropriate serial dilutions onto nutrient agar.
The count was expressed as log CFU/g.

Effect of the extracts on nucleic acid leakage from bacterial cells

The effect of acetone and ethyl acetate extracts on nucleic acid leakage (A260 nm) was
estimated as described earlier (20). To the 50 μl of the cell suspension (106 CFU), MIC of the
extracts was added, volume made upto 1 ml using PBS and incubated at 37 C. At different
time intervals [0 (immediately after addition of extracts), 15, 30 and 60 min], 50 μl of mixture
was added to 1.95 ml of PBS and absorbance was measured at 260 nm against PBS blank in a
UV-Visible spectrophotometer. For control, only cell suspension (50 μl) taken was added to
1.95 ml PBS and read as above. The leakage of nuclear material to incubating medium was
calculated in terms of A260 nm at each incubation period.

Effect of the extracts on bacterial cell wall

Scanning Electron Microscopy (SEM) was used to visualize the effect of extracts on
bacterial cell wall (21). Overnight cultures were centrifuged at 7000 rpm for 10 min at 4 C in

5
a refrigerated centrifuge (Plastocraft, Mumbai, India), washed twice with 0.1 M phosphate
buffer (pH 6.5) and volume was made up to 0.5 ml with same buffer. After addition of the
MIC of the extracts, final volume was made up to 1 ml using phosphate buffer. The above
cell suspension was incubated for 1 h and cells were harvested at 6000 rpm for 10 min at 4 ºC.
The pellet was incubated in 1 % glutaraldehyde at 0 ºC overnight and the cells were harvested
at 6000 rpm for 10 min at 4 ºC. The cells were dehydrated in ethanol gradient (10-100 %) and
coated with thin layer of gold using polaron SEM coating system. The cells were observed
with a LEO 435 VP Scanning Electron Microscope at 20 KV attached to Mitsubishi Video
copy processor. Photographs were taken using 35 mm Richo camera that was connected to
monitor optically through fibre optics.

Statistical analysis
All the experiments to evaluate antimicrobial efficacy of extracts in food systems and
leakage of 260 nm absorbing material were done in triplicate. Statistical analysis of the results
was done in Microsoft Excel. The t-test with an alpha level of 0.05 was used to compare the
differences in mean values of the control and treated samples.

Results
Effect of extracts on bacterial population in food model systems
The antibacterial effectiveness of Indian borage extracts was tested against E. coli, S.
aureus, B. cereus and Y. enterocolitica by artificially inoculating them (~ 5 log CFU) in
cabbage and papaya model systems (Figure 1 and 2). At the same time, the extracts were used
in chicken meat to study effect against natural microflora (Figure 3). In Cabbage food system,
2xMIC treatment of extracts resulted in lower viable counts as compared to controls for all the
bacteria. However, no definite trend was observed, as MIC and 2xMIC of both the extracts
significantly (p<0.05) lowered the counts of B. cereus, whereas only ethyl acetate extract at
2xMIC lowered counts of E. coli throughout the storage. MIC and 2xMIC of these extracts
showed significantly (p<0.05) lower count only at 7 days against S. aureus, and in 5 and 7
days against Y. enterocolitica. Almost similar trend for reduction in bacterial population was
observed in papaya food system albeit at a higher concentration. Both the extracts showed
significantly (p<0.05) lower count in papaya system for Y. enterocolitica and S. aureus
throughout the storage at 5xMIC, whereas 5xMIC of both the extracts showed significantly
lower bacterial count up to 3 days for B. cereus and up to 2 days for E. coli. There was no
significant difference in bacterial population in MIC and 2xMIC treated papaya samples (data

6
not presented for 2xMIC). In chicken meat, the count of natural microflora was reduced
significantly (p<0.05) by 5xMIC and 10xMIC of both the extracts during storage. No definite
trend was observed for reduction in bacterial counts by 5xMIC and 10xMIC of both the
extracts during storage. However, 10xMIC of both the extracts reduced the growth to
approximately 1 log CFU/g.

Effect of the extracts on nucleic acid leakage from bacterial cells

In the present study, the leakage of 260 nm absorbing material from bacterial cells
(Figure 4) was observed after treatment with the leaf extracts at the respective MIC of ethyl
acetate extract and acetone extract. The increase in A260 nm was 60-95% with ethyl acetate and
40-80% with acetone in comparison with control. The leakage observed was significantly
(p<0.05) higher for treated cells of B. cereus, Y. enterocolitica (except 60 min with acetone
extract) and S. aureus (except 30 and 60 min with both extract) as against that of control.
However, it was statistically (p<0.05) similar to control in case of E. coli treated cells. Both
the extracts caused statistically (p<0.05) similar leakage of 260 nm absorbing material in the
tested bacteria at all the time intervals, with exception of Y. enterocolitica, wherein ethyl
acetate extract caused statistically (p<0.05) higher leakage than acetone extract after 15 min
incubation (Figure 4).

Effect of the extracts on bacterial cell wall

Treatment with extracts caused disintegration of bacterial cell wall as could be
observed in the Scanning Electron Micrographs for both the extracts (Figure 5). MIC of
acetone extract was able to disintegrate the cell wall in B. cereus and Y. enterocolitica, but not
many changes were observed in the cell wall of E. coli and S. aureus. Untreated cells showed
a continuous thin smooth cell wall and loss of smoothness and uniformity was observed in
treated cells. The cell wall exhibited slimy appearance and rupture of cell wall was also
observed at MIC of extracts in B. cereus and Y. enterocolitica, whereas shrinkage of cells was
observed in S. aureus and E. coli.

Discussion
Effect of extracts on bacterial population in food model systems
In all the food systems (Figures 1, 2, 3), there was a marked increase in bacterial
population in control samples, whereas the bacterial numbers decreased after treatment with
extracts, with certain exceptions. However, the concentration of extract required varied with

7
complexity of food systems. It is reported that the inhibitory activity of the antimicrobial
extracts is lower in food systems as compared to in-vitro systems and differences in inhibitory
activity of antimicrobial compounds in different food model systems has been reported (22).
Penteado and Leitao (19) also observed that growth of Listeria monocytogenes was not
inhibited even at low temperature storage, although a decrease in count was observed at lower
pH. It is well known that the antimicrobial potency of antimicrobial compounds in food
systems is reduced when compared to in vitro conditions, as the presence of fats,
carbohydrates, proteins, salt and pH strongly influences the effectiveness of these agents (23,
24). Although chitosan, a natural preservative exerts bactericidal effect against bacteria in
model system, the microflora of apple juice was not inactivated by it, even at higher
concentrations (25). Studies on the effect of essential oils in fruit and vegetable products have
recorded the effect of foodstuffs on microbial resistance (26) and attributed it to the greater
availability of nutrients in foods compared to laboratory media, which may enable bacteria to
repair damaged cells faster (27).
The physical structure of a food may also limit the antibacterial efficacy of the
extracts. In the present study also, a higher resistance of microorganisms in papaya pulp
(Figure 2) as compared to the cabbage system (Figure 1) was observed. Gutierrez et al. (22)
evaluated the efficacy of essential oils of lemon balm, marjoram, oregano and thyme on food
model media based on lettuce, milk and meat. They reported that food composition and
structure have a significant effect on the growth of microorganisms. We also observed that, as
the complexity of a system increased, the extent of control of microbial population decreased
and higher amount of extract was required for the control of similar bacterial populations (in
cabbage, 2xMIC; papaya, 5xMIC; and chicken, 10xMIC; Figure 1, 2, 3). A similar trend of
reduction in bacterial count of chicken by seabuckthorn leaf extract was reported by Dhanze
et al. (28), wherein the meat was preserved for 7 days by 3% extract treatment as compared to
3 days by 1% extract and untreated meat spoiled after 1 day storage.

Effect of the extracts on nucleic acid leakage from bacterial cells

Earlier studies have indicated loss of 260 nm absorbing material as one of the killing
mechanism and suggested that increase in A260 nm in incubating medium may be due to the
death of the organism as it is the consequence of the loss of cytoplasm macromolecules (29,
30). In present study also, leakage of 260 nm absorbing material was observed and most of
the leakage occurred during initial period, followed by a slight increase with increase in
incubation period (Figure 4). Plant phenolics are known to interact with DNA (31, 32); and

8
Hammer et al. (33) observed significant loss of A260 nm absorbing material in Candida
albicans treated with Tea Tree oil. The leakage of A260 nm absorbing material from bacterial
cells by treatment with Indian borage extracts observed in present study may probably be due
to the alteration in cell membrane caused by the presence of phenolics (17). Stojkovic et al.
(30) also observed that protocatechuic acid, the main phenolic compound in Veronica
montana water extract inactivates L. monocytogenes by causing permeability changes in cell
membrane.

Effect of the extracts on bacterial cell wall

The antibacterial activity of various antimicrobials such as phenols, flavonoids,
terpenoids, coumarin and alkaloids present in natural preservatives is due to several
mechanisms, including cell wall disintegration and degradation of genetic material (12). In
present study, alteration in cell structure was observed in bacteria treated with Indian borage
extracts (Figure 5). Previously, Carson et al. (20) reported that the content of some cells of S.
aureus appeared depleted after treatment with Tea tree oil. In our study also, both the extracts
caused rupture of cells in B. cereus and Y. enterocolitica, but ethyl acetate extract showed
more structural alterations in S. aureus and E. coli. These findings are similar to earlier
reported observations of decrease in size of Listeria cells by essential oil (34), and structural
alterations induced by nitrofuran (31).

Conclusions
This study showed that Indian borage extracts have good antibacterial activity in
different food systems as they reduced the counts of artificially inoculated bacteria in model
systems and natural microflora in chicken meat. Probably, these extracts are causing the
leakage of cell constituents and degradation of bacterial cell wall, thereby decreasing the
population of bacteria. However, the concentration of extracts required for reducing counts
were high in food systems (2-10 fold of MIC observed in in-vitro system), therefore, the
effect of extract addition on sensory and textural properties of foods needs a deeper study.

Acknowledgements
Authors thank Director, CSIR-CFTRI, Mysore for the constant encouragement, and
Dr. M. C. Varadaraj, Chief Scientist, Department of Microbiology and Fermentation
Technology, CSIR-CFTRI, Mysore for critically going through the manuscript. The authors
wish to express sincere thanks to Mr. A. S. Chauhan, Principal Scientist, Fruit and Vegetable

9
Technology Department, CSIR-CFTRI, Mysore for identification of plant material, and Mr.
K. Anbalagan, Senior Technician, Central Instruments Facility and Services, CSIR-CFTRI,
Mysore for help in SEM analysis.

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 FIGURE CAPTIONS
Fig. 1. Effect of Indian borage leaf extracts on growth of bacteria in cabbage model system
(a- B. cereus; b- E. coli; c- S. aureus; d- Y. enterocolitica) (C- control; A- MIC of Acetone
extract; 2A- 2xMIC of Acetone extract; EA- MIC of Ethyl acetate extract; 2EA- 2xMIC of
Ethyl acetate extract)

Fig. 2. Effect of Indian borage leaf extracts on growth of bacteria in papaya pulp (a- B.
cereus; b- E. coli; c- S. aureus; d- Y. enterocolitica) (C- control; A- MIC of Acetone extract;
5A- 5xMIC of Acetone extract; EA- MIC of Ethyl acetate extract; 5EA- 5xMIC of Ethyl
acetate extract)

Fig. 3. Effect of various Indian borage leaf extract concentrations on growth of natural
microflora in chicken (C- control; 5A- 5xMIC of Acetone extract; 10A- 10xMIC of Acetone
extract; 5EA- 5xMIC of Ethyl acetate extract; 10EA- 10xMIC of Ethyl acetate extract)

Fig. 4. Effect of MIC of Indian borage leaf extracts on leakage of A260 nm absorbing material
from different bacteria (a- B. cereus, b- E. coli, c- S. aureus, d- Y. enterocolitica)

Fig. 5. Effect of MIC of Indian borage leaf extracts on B. cereus (a, b, c), E. coli (d, e, f), S.
aureus (g, h, i) and Y. enterocolitica (j, k, l) cell morphology

(a, d, g, j – untreated control; b, e, h, k – treated with MIC of acetone extract; c, f, i, l – treated

with MIC of ethyl acetate extract)

14
Fig. 1. Effect of Indian borage leaf extracts on growth of bacteria in cabbage model system
(a- B. cereus; b- E. coli; c- S. aureus; d- Y. enterocolitica) (C- control; A- MIC of Acetone
extract; 2A- 2xMIC of Acetone extract; EA- MIC of Ethyl acetate extract; 2EA- 2xMIC of
Ethyl acetate extract)

15
Fig. 2. Effect of Indian borage leaf extracts on growth of bacteria in papaya pulp (a- B.
cereus; b- E. coli; c- S. aureus; d- Y. enterocolitica) (C- control; A- MIC of Acetone extract;
5A- 5xMIC of Acetone extract; EA- MIC of Ethyl acetate extract; 5EA- 5xMIC of Ethyl
acetate extract)

16
Fig. 3. Effect of various Indian borage leaf extract concentrations on growth of natural
microflora in chicken (C- control; 5A- 5xMIC of Acetone extract; 10A- 10xMIC of Acetone
extract; 5EA- 5xMIC of Ethyl acetate extract; 10EA- 10xMIC of Ethyl acetate extract)

17
Fig. 4. Effect of MIC of Indian borage leaf extracts on leakage of A260 nm absorbing material
from different bacteria (a- B. cereus, b- E. coli, c- S. aureus, d- Y. enterocolitica)

18
 a b c

d e f

g h i

j k l

Fig. 5. Effect of MIC of Indian borage leaf extracts on B. cereus (a, b, c), E. coli (d, e, f), S.
aureus (g, h, i) and Y. enterocolitica (j, k, l) cell morphology

(a, d, g, j – untreated control; b, e, h, k – treated with MIC of acetone extract; c, f, i, l – treated

with MIC of ethyl acetate extract)

19